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Method Article

Stable Isotope In-Vivo Labeling for Mass-Spectrometry Identification of Paternal Metabolites Transferred from Sperm to Oocyte During Fertilization

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DOI:

10.3791/67765

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June 17th, 2025

 ,  ,  ,  , 
1Biogem Scarl, Institute of Molecular Biology and Genetics Research, 2Department of Experimental Medicine, University della Campania Luigi Vanvitelli, 3Department of Biology, University of Naples "Federico II ", 4Department of Pharmacy, School of Pharmacy, University of Salerno, 5Department of Pharmacy, University of Naples "Federico II "

In This Article

Summary

This procedure aims to identify metabolites transferred from sperm to oocytes during fertilization by using stable isotope labeling with 2H2O or U13C Glucose followed by metabolomic analysis. Deuterium-labeled sires are mated with unlabeled dams. Collected pre-implantation embryos are analyzed by mass spectrometry to sort paternal (deuterated) from maternal (non-deuterated) metabolites.

Abstract

Paternal contribution to embryo genetics has so far been limited to allelic sequences for decades. A decade of results suggests, instead, that epigenetic factors-DNA methylation, histone modifications, chromosomal organization, and regulatory RNAs-play crucial roles in paternal inheritance and influence embryonic development and zygote gene expression. Together with nucleic acids, sperm metabolome (lipids, carbohydrates, free amino acids) may act as epigenetic signal. This protocol aims to identify sperm-to-oocyte transferred metabolites by mass spectrometry. The procedure includes stable isotope labeling of sperm cells with 2H2O or U13C Glucose to trace, by metabolomics, paternal metabolites transferred to the oocyte during fertilization. The overall goal of the protocol is to reveal the role of paternal sperm metabolome in offspring's susceptibility to dysmetabolism, and it may be adapted to provide insights into how environmental conditions, such as diet and exposure to pollutants, alter paternal metabolic messages, potentially affecting offspring's health and predisposition to diabetes, obesity, and cardiovascular diseases.

Introduction

Paternal epigenome influences offspring's susceptibility to dysmetabolism
Differently from the plethora of pre- and post-conceptional roles exploited by mothers during embryo development, the contribution of sperm cells has been thought for decades to be exclusively genetic. According to genetic inheritance theory, paternal contribution to the phenotypic variability of an individual (including its susceptibility to disease) had to be ascribed exclusively to the genetic sequence of paternally inherited allelic variants and to their interactions with those maternally inherited1. Nowadays, it is clear that alleles are not the only genetic messages transmitted by the father to his offspring during fertilization and that, instead, a multitude of epigenetic signals contribute to paternal inheritance2,3,4. Epigenetic signals so far identified include: (1) methyl groups covalently added to cytosines of the parental genome5,6,7, (2) post-translational modifications (acetylation, methylation) of histones associated with sperm chromosomes and delivered to the oocytes8, (3) the 3D organization of paternal chromosomes leading to intrachromosomal interaction (also known as topologically association domains, TAD) persisting in the zygote9,10, and (4) regulatory RNA molecules (miRNAs, tRNA-derived fragments, and piwi-interacting RNAs)11,12 as well as rRNAs13 and tRNAs14 released from sperm into oocyte during fertilization.

The paternal epigenome is an important vehicle of information for the progeny. Paternal information will influence the zygote, the embryo during its development, and, after birth, offspring's propensity to dysmetabolism and diseases. This non-Mendelian inheritance (referred to as intergenerational or transgenerational depending on whether it affects one or multiple consecutive generations), is considered by many authors one of the undercover contributing factors responsible for the drastic increase in dysmetabolic diseases occurring in developed Western countries15. Indeed, according to the PoHaD (Paternal Origin of Health and Disease) and DoHaD (Developmental Origin of Health and Disease) theories, the parental epigenetic contribution is one of the factors predisposing offspring to develop diabetes, obesity, and cardiovascular diseases in their adulthood16,17. Interestingly, the parental epigenome is exceptionally susceptible to environmental stimuli and can be influenced by metabolic conditions (such as drug use, dietary habits, and metabolic fitness)18 as well as strictly environmental parameters (such as exposure to pollutants, social relationships, and mental and relational stress)5,11. Each of the abovementioned stimuli can thus alter the epigenomes of germinal cells (sperm and oocytes) and influence future generations.

High-fat diet (HFD) consumption represents one of the most extensively studied environmental stimuli capable of altering paternal epigenome and leading to deleterious messages delivered to offspring19. This stimulus affects offspring phenotype (up to the third generation), who often display a greater propensity to develop dysmetabolism, regardless of their caloric intake. Offspring from father consuming HFD manifest growth retard, glucose intolerance, and insulin resistance in both mice and rats20,21. In humans, epigenetic inheritance has primarily been studied in generations born during and immediately after periods of famine or overfeeding22. In the Överkalix cohort, the food availability of paternal grandparents during their slow growth period was correlated with overall mortality risk, cardiovascular diseases, BMI, waist circumference, and fat mass of their grandchildren23. Recently, evidence of paternal intergenerational transmission has been confirmed in other cohorts24.

Paternal metabolites involved in intergenerational inheritance
Despite clinical and epidemiological evidence, the molecular details of paternal intergenerational inheritance remain unclear. At first, the identification of all molecular transducers of epigenetic information is still to be completed. Scientific evidence suggests that the nucleic component of spermatozoa, by itself, cannot explain the complexity of the intergeneration inheritance. Variations in DNA methylation have been indeed measured in the spermatozoa of mice and rats fed HFD, but in such a low percentage of the sperm population that this cannot account for the high penetrance of the dysmetabolic phenotype observed in the offspring25. On the other hand, the pool of signaling RNAs transferred from sperm to oocytes has a very short half-life and, although capable of influencing zygotic gene expression, seems unlikely to be maintained at effective concentrations post-fertilization, in the zygote in day-2, day-3 embryos, in morulae and in blastocysts. Thus, it seems evident that new molecular determinants must be involved in the intergenerational inheritance mechanism.

Along with nucleic acids, the sperm also contributes metabolomic components to the zygote, such as lipids, carbohydrates, and amino acids26,27,28,29. These metabolites, particularly those endowed with modulatory activities, could influence zygotic gene expression and embryonic development and thus might act as epigenetic signals30. Furthermore, similarly to nucleic acids, the concentration and the relative abundance of these metabolites in sperm can be influenced by environmental conditions experienced by the father and thus represent vehicles of information for the offspring28,30,31,32,33,34. Indeed, the sperm metabolome of mature spermatozoa has been shown to depend on the father's health status and be influenced by environmental factors such as diet, physical exercise, toxic substances, and endocrine disruptors.

Understanding the role of sperm metabolites in embryonic development is complicated by several technical limitations: (1) the size difference between the sperm and the oocyte, which results in a minimal metabolic mass contribution of the sperm for the zygote, and (2) the several metabolomic variations the sperm undergoes during its stages of production (spermatogenesis), epididymal maturation, ejaculation, and fusion with the oocyte.

Fluctuation of sperm metabolome during sperm maturation
In mammals, spermatozoa are produced in the testis through spermatogenesis, a spermatogonial stem cell-dependent process by which committed spermatogonia develop into primary spermatocytes that enter meiosis and produce round haploid spermatids. Morphogenesis of these cells into spermatozoa occurs through spermiogenesis, a massive morpho-functional remodeling of spermatids, from round cells to elongated/condensed/highly-specialized cells, corresponding to the last phase of spermatogenesis. However, the resulting spermatozoa are immotile and not yet capable of fertilization35. After spermiation, as spermatozoa are released from the rete testis into the epididymis, sperm cells interact with epithelial epididymal cells and engage with proteins and exosomal vesicles secreted by these cells (epididymosomes, rich in sphingomyelin and arachidonic acid). Passage through the epididymis enables the sperm to reach full maturity and, even if maintained in a pre-capacitated state, they acquire competence for motility36. During epididymal transit, the sperm membrane becomes more negatively charged and enriched in sphingomyelin. The sperm membrane becomes as well more fluid due to a progressive reduction in cholesterol and a concomitant increase in polyunsaturated fatty acids (mainly arachidonic, docosapentaenoic, and docosahexaenoic acids). Post-ejaculation, in the female genital tract, seminal fluid proteins and albumin promote sperm capacitation by promoting hyperpolarization and calcium influx to activate sperm motility. Capacitated sperms' plasma membrane presents further increased fluidity, low cholesterol-to-phospholipid ratio, and reduced sialic acid, GM1 ganglioside, and triglycerides content31,37.

Before encountering the oocyte, the sperm undergoes the acrosome reaction, a fusion of the acrosome (a Golgi-derived membranous organelle that covers the anterior part of the sperm nucleus and is formed during spermiogensis) with the sperm plasma membrane, that facilitates sperm plasma membrane reorganization and competence to penetrate of the zona pellucida (ZP), a matrix of glycoproteins surrounding the oocyte35,38. As the final step, hemifusion between the sperm and oocyte membrane, promoted by sperm proteins IZUMO1, SPACA6, and TMEM9539, results in the incorporation of sperm lipids into the oocyte membranes and the transfer of the nucleic and metabolic content into the oocyte.

Rationale of the procedure
The aim of this procedure is to identify metabolites transported by the sperm and transferred into the oocyte at fertilization (Figure 1). To distinguish between sperm- and oocyte-derived metabolites in the zygote, sperms are labeled with Deuterium (2H, a hydrogen isotope) or 13C (a carbon isotope) using in vivo metabolic labeling with Deuterium Oxide, 2H2O40, or uniformly labeled U13C Glucose41. Isotope-labeled male mice are then mated with unlabeled females to ultimately collect zygotes, morulae, and blastocysts. Early-stage embryos are processed to extract (1) polar, (2) non-polar metabolites, and (3) sterols. Metabolomes are then analyzed using mass spectrometry techniques. Isotopically-labeled (paternal) metabolites will be identified and distinguished from non-labeled (maternal) ones, unequivocally demonstrating their paternal origin.

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Protocol

All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Naples Federico II, Italy. The reagents and the equipment used in this study are listed in the Table of Materials.

1. Selection and grouping of C57BL/6J males

  1. Select C57BL/6J males of about 12 weeks.
  2. Divide males into two groups and name them LABELED ( undergoing stable isotope labeling) or UNLABELED (used as control and for measuring the efficacy of isotope labeling in sperm cells and morulae).
  3. In order to empty the epididymis from unlabeled sperm cells, allow the males to mate with paired females.

2. Stable isotopic labeling of C57BL/6J males

  1. If using 2H2O for in-vivo labeling, prepare 10% v/v 2H2O -enriched water to be used as drinking water by mixing 100 mL of 2H2O with 900 mL of tap water. If using U13C-Glucose for in-vivo labeling, prepare 5%-15% U13C-Glucose water to be used as drinking water by dissolving 50-150 g of U13C-Glucose in 1 L of tap water;
    CAUTION: Prolonged consumption of 2H2O is toxic for mice. Set for each specific mouse strain, the maximal amount of 2H2O to add in drinking water to achieve maximal labeling efficiency and minimal side effects. Prolonged consumption of Glucose causes hyperglycemia and diabetes in mice. Set the maximal amount of U13C-Glucose to add to drinking water to achieve maximal labeling efficiency and minimal side effects.
  2. Replace the water bottles with those containing isotope-labeled drinking water and allow mice to drink it for 2.5-5 weeks (covering at least half spermatogenesis), renewing the liquid twice a week.
  3. At the end of the treatment period, allow the labeled males to mate with superovulated females.

3. Superovulation of C57BL/6J females

  1. Select females of about 4 weeks with a weight between 12.5-14 g, in prepubertal age.
  2. Induce the follicle maturation by intraperitoneal injection of 5 IU/50 µL/mouse of PMSG (Pregnant mare's serum).
  3. After 47 h, inject intraperitoneally of luteinizing hormone HCG (Human chorionic gonadotropin, 5 IU/50 µL/mouse) to promote completion of the maturation of the follicle and induce ovulation and the formation of the corpus luteum.
  4. After the HCG injection, allow the females to mate with LABELED and UNLABELED mice, according to step 1 of Figure 1.
  5. After mating, perform the vaginal plug control42. Separate the positive females and cage them.

4. Pre-implantation embryo collection by flushing

  1. Sacrifice vaginal plug-positive females following institutionally approved protocols (2.5 Day Post Coitum to collect morulae).
  2. Place the animals in dorsal recumbency on the table; wet the abdomen with 70% ethanol, and perform a wide laparotomy.
  3. Remove the uteri on one side by pinching the cervix and cutting at the caudal end of the cervix; on the other side, by cutting at the end of the uterine horn (infundibular portion).
  4. Place the uteri in the Petri dish with PBS to rinse them and then transfer them to another clean Petri dish to avoid the presence of residues.
  5. In order to flush the uteri, load a 2.5 mL syringe with the culture medium and attach it to a 33 G blunt-tip Hamilton needle.
  6. Insert the needle into the infundibulum of the oviduct prepared as described above and wash the entire uterus.
  7. Collect the morulae in a drop of culture medium to wash them from any residual mucosa and blood.
  8. Transfer collected samples in new collection tubes, and group them in LABELED MORULAE and UNLABELED MORULAE, depending on whether they derive from mating events involving LABELED or UNLABELED sires. Snap-freeze the samples in liquid nitrogen.
  9. Sacrifice a group of unmated superovulated females (following institutionally approved protocols) in order to collect unfertilized oocytes. Transfer the collected samples to new collection tubes, and label them as UNLABELED OOCYTES. Snap-freeze the samples in liquid nitrogen.

5. Sperm collection from epididymal cauda

  1. Sacrifice LABELED or UNLABELED sires.
  2. Place the animals in dorsal recumbency on the table, wet the abdomen with 70% ethanol, and perform a wide laparotomy.
  3. Remove the epididymis and immediately immerse them in 3 mL of PBS (pH 7.6) and loosely cut them to drain sperm from the ducts.
  4. Filter the sperm samples through the cheesecloth.
  5. Centrifuge the sperm cells at 800 x g for 10 min (at room temperature).
  6. Resuspend the pellet in 1 mL of cold deionized water. Only sperm cells will resist the hypotonic solution, while non-sperm cells will burst.
  7. Centrifuge sperm cells at 800 x g for 10 min at room temperature.
  8. Label the tubes, and group them as LABELED SPERM and UNLABELED SPERM, depending on whether they were collected from LABELED or UNLABELED sires.
  9. Snap freeze the samples in liquid nitrogen and store samples at -80 °C.

6. Extraction of lipids and polar metabolites

  1. Equilibrate the sample from steps 1-5 at 4° C on ice.
  2. Add 225 µL of ice-cold methanol (MeOH) to the samples and incubate at -30 °C for 1 min and, afterward, put them in a sonic bath for 10 min.
    1. Optional: while setting the experimental condition, add 0.1-2 ppm of both C16-Ceramide-13C3-15N and L-Lysine-ε-15N hydrochloride as internal standards to measure the efficiency of extraction for non-polar and polar metabolites, respectively.
  3. Add 750 µL of ice-cold methyl tert-butyl ether (MTBE) in a Thermomixer for 1 h at 4 °C (550 rpm).
  4. Add 188 µL of H2O to the samples after centrifuging them. Separate the upper phases (lipids) from the lower phase (polar metabolites) and dry them.
    CAUTION: MeOH causes blindness and damage to the liver, kidneys, and heart if swallowed. Overexposure has an accumulative effect on the central nervous system. MTBE may cause skin, eye, and respiratory irritation. Work under a fume hood.
  5. Dry the sample using a vacuum concentrator. Store dried samples at -80 °C until mass/spectrometry analysis.
    NOTE: Dried samples can be stored at -80 °C for up to 6 months, waiting for mass/spectrometry analysis. For the identification of metabolites less represented in pre-implantation embryos, it might require pooling samples obtained from the same mating pair and mating event.

7. Extraction of sterols

NOTE: Avoid the use of plastic tubes and tips [they will be dissolved by the non-polar organic solvent]; instead, use glass tubes and glass Pasteur pipettes to dispense liquids.

  1. Equilibrate the sample from steps 1-5 at 4 °C on ice. Resuspend the collected samples in 500 µL of a solution of Methanol/H20 70% v/v and transfer the suspension to a glass tube.
    1. Homogenize by vortexing for 30 s. Leave the homogenate for 16 h at 4 °C.
    2. Optional: while setting experimental condition, add 0.1-2 ppm of the plant sterol Sitostanol-5,6,22,23-d4 as internal standards to measure the efficiency of extraction for sterols.
      CAUTION: MeOH causes blindness and damage to the liver, kidneys, and heart if swallowed. Overexposure has an accumulative effect on the central nervous system.
  2. Centrifuge the sample at 3.220 x g for 15 min at room temperature. Transfer the supernatant to a clean glass tube and supplement it with 500 µL of PBS.
  3. Add 7 mL of di ice-cold Diethyl ether and vortex for 1 min. Let the sample stand for 1-2 min. and wait for phase separation. Freeze by submerging the tube in a dry ice/acetone bath. Transfer the unfrozen Diethyl ether phase to a newly labeled glass tube.
    CAUTION: Diethyl ether is an extremely flammable liquid and vapor and decomposes to explosive peroxides in air and light. Do not work in close contact with flames. Diethyl ether is dangerous if swallowed and harmful to the skin.
  4. Defrost the water phase at 37 °C and repeat step 4.3. Combine Diethyl ether phases collected at steps 4.3-4.4 in a single glass tube.
  5. Dry the sample using a vacuum concentrator. Store dried samples at -80° C until mass/spectrometry analysis.

8. Sample derivatization

NOTE: Samples would benefit from TMS derivatization for the analysis of specific metabolites (cholesterol, fatty acids, amino acids).

  1. Equilibrate the samples at room temperature
    CAUTION: Incomplete equilibration may cause condensation of atmospheric water on tube walls and samples. Water severely competes for TMS derivatization.
  2. Solubilize the samples in 50 µL of pyridine and derivatize them by adding 25 µL of N,O-Bis(trimethylsilyl(TMS)trifluoroacetamide (BSTFA). Incubate the samples for 90 min at 60 °C. Spin the samples and immediately proceed with GC-MS analysis.
    CAUTION: Pyridine is flammable, irritant, and toxic. BSTFA is flammable, corrosive, and irritant. Work under a fume hood and use protective gloves.

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Results

In order to monitor metabolite transfer from sperm to oocyte, morulae isolated from C57 BL/6J females mated with sires from LABELED and UNLABELED groups are collected and analyzed by Mass-Spectrometry based Metabolomics. Metabolites were extracted as described in steps 6-7 and derivatized with TMS as described in step 8. Metabolomic and Lipidomic profiling of LABELED morulae, UNLABELED morulae, UNLABELED oocytes, LABELED, and UNLABELED sperm cells was performed. The resulting profiles were statistically analyzed, aligned...

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Discussion

The described protocol allows the identification of metabolites transferred from sperm to oocytes during fertilization by using stable isotope labeling of male mice with 2H2O or U13C Glucose followed by metabolomic analysis. Isotopically labeled metabolites found in morulae are of paternal origin.

Although hydrogen and deuterium are isotopes, they are not entirely identical in terms of chemical behavior. The melting and boiling points of 2H2

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Disclosures

The authors declare no competing interests.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
2H2O filtered, 99.8 atom % 2HSigma Aldrich756822
AcetoneSigma Aldrich179124
Agilent  SIL-8Agilent (Santa Clara, CA, U.S.A.)
C16-Ceramide-13C3-15NLGC standardTRC-C342770-25MG
dichloromethaneSigma Aldrich34856
Diethyl ether Sigma Aldrich673811
ethanolSigma Aldrich1,11,727
HCG (Human chorionic gonadotropin,Sigma AldrichCG5-1VL
L-Lysine-ε-15N hydrochlorideSigma Aldrich608971
Male and female  C57BL/6JOlaHsd miceInotiv C57BL/6JOlaHsd
methyl tert-butyl ether (MTBE)Sigma Aldrich306975
MetOHSigma Aldrich1,06,035
N,O-Bis(trimethylsilyl(TMS)
trifluoroacetamide (BSTFA)		Supelco15238
PBSApplichemA0965-9010
PMSG (Pregnant mare’s serum)Lee Biosolutions493-10
pyridineSigma Aldrich360570
Shimadzu GCMS 2010plus Shimadzu
Sitostanol-5,6,22,23-d4Sigma Aldrich680028
sodium trifluoracetateSigma Aldrich1615138
SolariX XR 7T Bruker Daltonics
TimsTOF Pro Q-TOF Bruker Daltonics
U13C glucose Sigma Aldrich389374
Ultimate RS3000 UHPLC Thermo Fisher Scientific
VanGuard CSHTM precolumn (5.0 × 2.1 mm; 1.7 μm, 130 Å)Waters

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